This invention relates to methods and compositions for manipulating bacterial resistance to non-antibiotic antibacterial compositions, disinfectants and organic solvents.
Antibiotic or antimicrobial substances have long been used to inhibit the growth of bacteria or other microbes and to treat bacterial or microbial infections in humans, other animals, and in tissue culture. The use of antibiotics or antimicrobials in a treatment regimen, however, has the undesirable effect of selecting for bacteria or other microbes which are resistant to those antibiotics or antimicrobials which are administered or applied. As a result, treatment regimens can be adversely affected or, in some cases, rendered ineffective. This necessitates a continual search for new antibiotics and antimicrobials.
Of particular interest is the discovery of bacteria which express a multiple antibiotic resistance phenotype (Mar). This phenotype entails simultaneous resistance to a multiplicity of antibiotics which are unrelated in chemical structure. The appearance of such bacteria and infections by such bacteria greatly increase the difficult) of identifying effective antibiotics and treating infections in humans or other animals.
Multiple antibiotic resistance in bacteria is most commonly associated with the presence of plasmids and/or transposons which contain one or more resistance genes, each encoding a single antibiotic resistance phenotype. Multiple antibiotic resistance associated with the chromosome, however, has been reported in Klebsiella, Enterobacter, Serratia (Gutmann et al., J. Infect. Dis. 151:501-507, 1985), Neisseria (Johnson and Morse, Sex. Transm. Dis. 15:217-224, 1988), and Escherichia (George and Levy, J. Bacteriol. 155:531-540, 1983).
Bacteria expressing a chromosomal multiple antibiotic resistance phenotype can be isolated by selecting bacteria with a single antibiotic and then screening for cross-resistance to structurally unrelated antibiotics. For example, George and Levy initially described a chromosomal multiple antibiotic resistance system which exists in Escherichia coli and which can be selected by a single drug, e.g., tetracycline or chloramphenicol (George and Levy, 1983). In addition to resistance to the selective agents, the Mar phenotype includes resistance to structurally unrelated agents, including nalidixic acid, rifampin, penicillins, and cephalosporins (George and Levy 1983) as well as fluoroquinolones (Cohen et al. 1989).
The chromosomal gene locus which correlates with the Mar phenotype observed in E. coli has been identified. The chromosomal mar locus, located at 34 min on the E. coli chromosomal map, is involved in the regulation of intrinsic susceptibility to structurally unrelated antibiotics (Cohen et al., J. Bacteriol. 175:1484-1492, 1993; Cohen et al., Antimicrob. Agents and Chemother. 33:1318-1325, 1989; Cohen et al., J. Bacteriol. 170:5416-22, 1988; Goldman et al., Antimicrob. Agents Chemother. 40:1266-1269, 1996), as well as the expression of antioxidant genes (Ariza et al., J. Bacteriol. 176:143-148, 1994; Greenberg et al., J. Bacteriol. 173:4433-4439, 1991) and internal pH homeostasis (Rosner and Slonczewski, J. Bacteriol. 170:5416-22, 1994). The mar locus consists of two transcription units (marC and marRAB) which are divergently transcribed from a central putative operator-promotor region (marO) (Cohen et al., 1993; Goldman et al., 1996). marR is the repressor of the marRAB operon (Cohen et al., 1993; Martin and Rosner, Proc. Natl. Acad. Sci. USA 92:5465-5460, 1995; Seoane and Levy, J Bacteriol. 177:3414-3419, 1995). Mutations in marR result in increased expression of the marRAB operon. Overexpression of marA alone is sufficient to produce the multiple antibiotic resistance phenotype (Cohen et al., 1993; Gambino et al., J. Bacteriol. 175:2888-2894, 1993; Yan et al., Abstr. A-26, p. 5, In Abstracts of the 1992 General Meeting of the American Society for Microbiology, American Society for Microbiology, Washington, DC, 1992). marB has no effect of its own; however, when it is present on the same construct with marA, it produces a small increase in antibiotic resistance (White et al., Abst A-104, p. 20. In Abstracts of the 1994 General Meeting of the American Society for Microbiology, American Society for Microbiology, Washington, D.C. 1994). The function of marC is unknown; however, it also appears to enhance the multiple antibiotic resistance phenotype when cloned on the same DNA fragment with the marRAB operon (Goldman et al., 1996; White et al., 1994).
Overexpression of marA confers multiple antibiotic resistance via increased efflux of antibiotics, including fluoroquinolones, tetracycline, and chloramphenicol (Cohen et al., 1989; George and Levy, 1983; McMurry et al., Antimicrob. Agents Chemother. 38:542-546, 1994). Transcription of the acrAB operon, which encodes a multi-drug efflux pump whose expression is modulated by global stress signals (Ma et al., Mol. Microbiol. 16:45-55, 1995; Ma et al., Mol. Microbiol. 19:101-112, 1996), was shown to be elevated in strains containing marR mutations and displaying the Mar phenotype (Okusu et al., J. Bacteriol. 178:306-308, 1996). Moreover inactivation of acrAB led to increased antibiotic susceptibility in wild type and Mar mutants (Okusu et al., 1996).
More recently, mutations of marR have been found in clinical isolates resistant to quinolones (Maneewannakul and Levy, 1996). Thus mar mutants can be selected under clinical conditions and not merely under controlled laboratory conditions. Early mar mutants (i.e., xe2x80x9cfirst-stepxe2x80x9d mar mutants) remain susceptible to many common antibiotics, although such mutants can achieve levels of clinical resistance to certain antibiotics, including tetracycline, nalidixic acid and rifampin (reviewed by Alekshun and Levy, Antimicrob. Agents Chemother. 41:2067-2075, 1997). First-step mar mutants thus may serve as precursors of bacterial mutants which display higher levels of resistance resulting from additional mutations on the chromosome. Thus it has been demonstrated that antibiotics can select for mutations in chromosomal gene loci which confer multiple antibiotic resistance under clinical conditions.
Non-antibiotic antibacterial compositions such as disinfectants are widely used in both clinical and consumer environments for reducing bacterial contamination of work surfaces, equipment, products and the like. These non-antibiotic antibacterial compositions have been incorporated into a wide spectrum of cleansers, disinfectant compositions, soaps, lotions, plastics, etc. It is not known whether exposure of bacteria to non-antibiotic antibacterial compositions also can select for bacterial mutants , including those which display a multiple antibiotic resistance phenotype.
It has now been discovered that bacterial mutants having multiple antibiotic resistance can be selected by non-antibiotic antibacterial agents such as common disinfectants. It further has been discovered that the phenotype of the multiple antibiotic resistant mutants selected by a non-antibiotic antibacterial agent results from mutations in chromosomal gene loci which regulate expression of efflux pumps, which loci have been implicated in multiple antibiotic resistance phenotypes as described above. The efflux pumps actively pump out the non-antibiotic antibacterial agents, as well as organic solvents and antibiotics, thereby rendering the mutant bacteria resistant to all of the foregoing compounds.
According to one aspect of the invention, a method is provided for inhibiting the selection and/or propagation of a bacterial mutant that overexpresses an efflux pump. Bacteria are contacted with an agent that binds to a gene locus (the expression of the gene locus enhances expression of the efflux pump) or an expression product thereof, in an amount effective to inhibit the gene locus-enhanced expression of the efflux pump. In preferred embodiments, the gene locus is selected from the group consisting of a mar locus, a sox locus and a rob locus. Also in preferred embodiments, the efflux pump is acr-like, including the acrAB efflux pump.
The agent can be selected from the group consisting of chemicals, antisense nucleic acids, antibodies, ribozymes, and proteins which repress expression of the gene locus. A preferred embodiment is an agent that is an antisense nucleic acid, and in particularly preferred embodiments, the agent is antisense to the mar locus, sox locus and/or rob locus. Another preferred embodiment is chemical inhibitors of efflux pumps, particularly L-phenylalanyl-L-arginyl-xcex2-naphthylarnide.
According to another aspect of the invention, a method is provided for rendering bacterial cells more susceptible to a non-antibiotic bactericidal or bacteriostatic agent that is a substrate of an efflux pump. An inhibitor of a gene locus or an expression product thereof is administered to a bacterial cell, wherein the expression of the gene locus enhances expression of an efflux pump. In preferred embodiments the gene locus is selected from the group consisting of a mar locus, a sox locus and a rob locus. In other preferred embodiments the efflux pump is acr-like and can be acrAB. The preferred inhibitors are as described above.
According to still another aspect of the invention, a method is provided for rendering bacterial cells more susceptible to a non-antibiotic bactericidal or bacteriostatic agent that is a substrate of an efflux pump. The method involves administering to the bacterial cell an inhibitor of the efflux pump. In preferred embodiments the efflux pump is acr-like and can be acrAB. Preferably the inhibitor is selected from the group consisting of about 4% ethanol, methanol, hexane, minocycline and L-phenylalanyl-L-arginyl-xcex2-naphthylamide.
According to another aspect of the invention, a method is provided for modulating (increasing or decreasing) the ability of bacterial cells to survive in an organic solvent. In certain embodiments the method involves enhancing expression in the bacterial cells of an organic solvent bacterial efflux pump by growing the bacterial cells in the presence of a non-mar/sox/rob inducing agent, wherein the agent induces the overexpression of the organic solvent bacterial efflux pump. The agent can be a gene encoding an acr-like pump, the acrAB pump, or expression products thereof. In other embodiments the method involves reducing expression in the bacterial cells of an organic solvent bacterial efflux pump by growing the bacterial cells in the presence of an agent, wherein the agent reduces the expression of the organic solvent bacterial efflux pump. The agent can be an antisense nucleic acid which binds to a gene locus encoding an acr-like pump, especially the acrAB pump, a gene locus which enhances expression of an efflux pump, such as marA, soxA and robA, and the like. The agent also can be a ribozyme or a protein which represses expression of the gene locus. The agent also can be an antibody to an expression product of the foregoing genes. The agent also can be a chemical compound which reduces expression of the efflux pump, or reduces activity of the efflux pump, such as L-phenylalanyl-L-arginyl-xcex2-naphthylamide.
According to another aspect of the invention, a method is provided for testing the ability of a non-antibiotic composition to induce a multiple antibiotic resistance phenotype in a bacterium. The bacterium is contacted with the non-antibiotic composition. The expression of a bacterial gene locus is determined, the altered expression of which is indicative of induction of the multiple antibiotic resistance phenotype in the bacterium. Then, the result of this determination is compared with a control, wherein altered expression of the bacterial gene locus indicates that the non-antibiotic composition induces the multiple antibiotic resistance phenotype in the bacterium. In preferred embodiments, the gene locus is selected from the group consisting of a mar locus, a sox locus, a rob locus and an acr-like efflux pump locus. In one particular embodiment the efflux pump locus is acrAB. The foregoing methods can be carried out using a non-antibiotic composition that is an inactive ingredient. The inactive ingredient can be a non-bactericidal ingredient. The inactive ingredient also can be a non-bacteriostatic ingredient. In one preferred embodiment the method is carried out by determining the enzymatic activity of an expression product of a marker gene, preferably lacZ, fused to the bacterial gene locus.
According to another aspect of the invention, a composition is provided. The composition includes a non-antibiotic bactericidal or bacteriostatic first agent and a second agent that inhibits the expression of activity of an efflux pump. In one embodiment, the second agent inhibits the expression of a gene locus or an expression product thereof, wherein the expression of the gene locus enhances expression of the efflux pump. In preferred embodiments, the second agent is selected from the group consisting of antisense nucleic acids, antibodies, ribozymes and proteins that repress expression of the gene locus. In one preferred embodiment the second agent inhibits an acr-like efflux pump, and particularly preferred is an antisense nucleic acid. The second agent also can be selected from the group consisting of 4% ethanol, methanol, hexane, minocycline and L-phenylalanyl-L-arginyl-xcex2-naphthylamide. The preferred second agent is L-phenylalanyl-L-arginyl-xcex2-naphthylamide. The first agent in some embodiments is selected from the group consisting of triclosan, pine oil, quaternary amine compounds including alkyl dimethyl benzyl ammonium chloride, chloroxylenol, triclocarbon, disinfectants and organic solvents.
According to still another aspect of the invention, a method for identifying an antibacterial composition which does not select for or induce a multiple antibiotic resistance phenotype in a bacterium is provided. The bacterium is contacted with the antibacterial composition. The expression of a bacterial gene locus is determined, the altered expression of which is indicative of induction of the multiple antibiotic resistance phenotype in the bacterium. Then, the result of this determination is compared with a control, wherein altered expression of the bacterial gene locus indicates that the antibacterial composition induces the multiple antibiotic resistance phenotype in the bacterium and a lack of altered expression of the bacterial gene locus indicates that the antibacterial composition does not induce the multiple antibiotic resistance phenotype in the bacterium. In preferred embodiments, the gene locus is selected from the group consisting of a mar locus, a sox locus, a rob locus and an acr-like efflux pump locus. In one particular embodiment the efflux pump locus is acrAB. In one preferred embodiment the method is carried out by determining the enzymatic activity of an expression product of a marker gene, preferably lacZ, fused to the bacterial gene locus.
The invention also provides methods for identifying antibacterials which are not subject to efflux pumps, e.g. those antibacterials which are not substrates for efflux pumps. These antibacterials are those which have bactericidal or bacteriostatic action against bacteria which express an efflux pump, particularly those which overexpress an efflux pump, particularly an acr-like pump, especially acrAB. These and other aspects of the invention are described in greater detail below.